Epidemiological studies have linked fibrinogen and fibrin clot structure to most, if not all, thrombotic diseases. Our proposed studies are designed to determine the biochemical basis for this connection. Investigators have examined the conversion of fibrinogen to fibrin and the factors that control fibrin clot structure for over 50 years, leading to a substantial body of knowledge. Nevertheless, many critical issues remain ill defined. In particular, the specific interactions that control FXIII activation and lateral aggregation have not been identified. Our goal is to identify these interactions. We will use two approaches to determine the interactions that control FXIII activation, which occurs on the surface of a fibrin clot. First, we will use specific variant fibrinogens as substrates and follow the kinetics of FXIII activation. If the specific variants inhibit interactions between FXIII and fibrin and thrombin, the enzyme that activates FXIII, then we will see changes in the kinetics. Second, we will use crosslinking reagents to covalently link FXIII to fibrinogen, or fibrin, along with thrombin. We will cleave the crosslinked products into small peptides and determine their structures using mass spectrometry. These studies will define the fibrin specific interactions that promote FXIII activation and characterize the mechanisms that mediate this fibrin function. Lateral aggregation is the last step in the conversion of soluble fibrinogen into an insoluble fibrin polymer. Similar to our FXIII studies, we will use two approaches to determine the molecular interactions that participate in this step. First, we will synthesize variant fibrinogens which specific residue changes in regions thought to be critical for lateral aggregation. We will use kinetic studies to determine whether and how these substitutions influence the rate of lateral aggregation. We will use electron microscopy to examine the associated changes in fibrin structure. Second, we will determine the fibrin residues that are juxtaposed in a fibrin polymer using chemical crosslinking reagents to form covalent bonds between the individual fibrin units. We will cleave the crosslinked products into small peptides and determine their structures using mass spectrometry. We will map these structures to the surfaces of fibrin to locate the juxtaposed regions. These two studies will define the interactions that occur in lateral aggregation and characterize their relative roles. Data obtained from the proposed in vitro studies will lead to a more complete understanding of the events that are critical for effective clot formation in vivo. This understanding will likely enhance our ability to diagnose and treat thrombotic disease. PUBLIC HEALTH RELEVANCE: Epidemiological studies have linked fibrinogen and fibrin clot structure to most, if not all, thrombotic diseases-coronary artery disease, ischemic heart disease, stroke and thromboembolic disease. Our studies are designed to determine the mechanisms that control fibrin clot structure. Data obtained from these studies will lead to a more complete understanding of the events that are critical for an effective clot structure, which will likely improve our ability to diagnose and treat thrombotic disease.